RESUMO
In this study, a high amount of bioactive recombinant canine interferon-alpha subtype 4 (CaIFN-alpha4) was expressed in a baculovirus system. For easy purification, it was expressed as a CaIFN-alpha4 bearing histidine hexamer at the C-terminal region, designated CaIFN-alpha4His. CaIFN-alpha4His was detected in culture supernatants of insect cells infected with the recombinant virus using sodium dodecyl sulfate-polyarcylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue staining. The level of expression was very high, and approximately 1 mg of purified protein, with 5.0 x 10(7) units/mg, was obtained from 300 ml of culture supernatant. The purified product showed antiviral activity against Vesicular stomatitis virus on canine tumor cell line A72 and chicken embryo fibroblast cells.
Assuntos
Cães , Interferon-alfa/metabolismo , Animais , Baculoviridae , Linhagem Celular , Regulação da Expressão Gênica , InsetosRESUMO
We cloned five new subtypes of cDNA encoding canine interferon-alpha (CaIFN-alpha) from a canine epithelial cell line. CaIFN-alphas were divided into two groups by amino acid sequences and a molecular phylogenic tree. Two subtypes of them were expressed in Escherichia coli, and IFN proteins were purified. Recombinant CaIFN-alphas were highly species-specific and showed antiviral activity against Vesicular stomatitis New Jersey virus and canine adenovirus-1 , but not against canine herpesvirus-1.
Assuntos
Clonagem Molecular/métodos , Cães/genética , Expressão Gênica , Interferon-alfa/genética , Filogenia , Adenovirus Caninos , Sequência de Aminoácidos , Animais , Linhagem Celular , Efeito Citopatogênico Viral , Primers do DNA , DNA Complementar/genética , Escherichia coli , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência , Especificidade da Espécie , VesiculovirusRESUMO
Two kinds of FeIFN-alpha consisting of 166 amino acids (aa) and 171 aa were expressed in Escherichia coli, and the purified proteins were tested for antiviral activity on homologous and heterologous animal cells. Crude FeIFN induced in feline cells revealed antiviral activity on both homologous and heterologous animal cells. In contrast, both types of recombinant FeIFN-alpha revealed antiviral activity only on the feline cells. All of the FeIFN-alpha subtypes showed high activity to vesicular stomatitis virus, and the three species of feline viruses belonging to different families.
Assuntos
Gatos/imunologia , Interferon-alfa/biossíntese , Interferon-alfa/fisiologia , Vírus/imunologia , Animais , Calicivirus Felino/imunologia , Gatos/virologia , Escherichia coli , Peritonite Infecciosa Felina/imunologia , Expressão Gênica , Herpesviridae/imunologia , Organismos Geneticamente Modificados , Especificidade da Espécie , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
Mammalian interferon (IFN)-alpha consists of a 23-amino acid signal peptide and a 166-amino acid mature protein. Feline (Fe) IFN-alpha has an extra unique molecule consisting of a 171-amino acid mature protein with a 5-amino acid insertion. We cloned eight new subtypes of cDNA encoding FeIFN- alpha from a feline epithelial cell line. Among all the FeIFN-alpha subtypes, including six that have previously been reported, the variations were found to be far less than those of IFN-alphas of other animals.
Assuntos
Gatos/genética , DNA Complementar/genética , Interferon-alfa/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Primers do DNA , Células Epiteliais , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Homologia de Sequência de AminoácidosRESUMO
A rounding effect was demonstrated in cultured cells inoculated with the culture filtrates (CFs) of 60 strains of Staphylococcus intermedius derived from dogs affected with pyoderma. Exfoliative toxin (ET)-like toxin (ETLT) was isolated from the CF of S. intermedius strain D-52, which exhibited strong rounding activity and then was purified by gel filtration on a Sephadex G-75 column, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The ETLT caused exfoliation in 1-day-old chickens, suckling Syrian hamsters, and dogs, but not in suckling mice. The ETLT was serologically different from exfoliative toxin A (ETA), exfoliative toxin B (ETB), exfoliative toxin C (ETC), S. hyicus exfoliative toxin A (SHETA), and SHETB, as shown by Western blot analysis. The molecular weight of the ETLT was estimated at 30 kDa by SDS-PAGE. In the present study, we propose the ETLT was a novel type of ET, S. intermedius exfoliative toxin (SIET).
Assuntos
Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/toxicidade , Doenças do Cão/microbiologia , Exfoliatinas/isolamento & purificação , Exfoliatinas/toxicidade , Pioderma/veterinária , Infecções Cutâneas Estafilocócicas/veterinária , Staphylococcus/metabolismo , Animais , Anticorpos Antibacterianos/metabolismo , Western Blotting/veterinária , Galinhas , Cromatografia em Gel/veterinária , Cricetinae , Cães , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Pioderma/microbiologia , Ratos , Organismos Livres de Patógenos Específicos , Infecções Cutâneas Estafilocócicas/metabolismo , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus/químicaRESUMO
An exfoliative toxin (SIET)-producing strain (D-52) of Staphylococcus intermedius derived from canine pyoderma did not possess large plasmids. Therefore, the gene coding for SIET was considered to be located on the chromosomal DNA. The SIET gene was cloned from the chromosomal DNA of S. intermedius and was expressed in Escherichia coli. The nucleotide sequence of the SIET gene consists of a coding region of 990 bp specifying a polypeptide of 330 amino acid residues, which included a putative 42-residue signal sequence.